Prolines as antimicrobial agents

ABSTRACT

Transfer ribonucleic acid (tRNA) synthetase inhibitors, salts, and pharmaceutically acceptable compositions thereof of the general formula:                    
     wherein Ar is aryl and heteroaryl; L is —C(O)N(Q)CH 2 —, or —CR 10 R 11 OCR 12 R 13 —; Q is hydrido, —(CH 2 ) m CO 2 H and —(CH 2 ) m CO 2 CH 3 , m is 1, 2, 3, and 4; R 1 , R 2 , R 9 , R 10 , R 11 , R 12  and R 13  are hydrido or lower alkyl; wherein Het is a heterocyclic moiety, the inhibitors are suitable for use as antimicrobial agents.

RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application,Ser. No. 60/132,546, filed on May 5, 1999.

FIELD OF THE INVENTION

This invention relates to the field of transfer ribonucleic acid (tRNA)synthetase inhibitors, their preparation and their use as antimicrobialagents.

BACKGROUND OF THE INVENTION

Aminoacyl tRNA synthetases (aaRS) are a family of essential enzymes thatare found in virtually every biological cell and are responsible formaintaining the fidelity of protein synthesis. They specificallycatalyze the aminoacylation of tRNA in a two step reaction:

amino acid (AA)+ATP=>AA-AMP+PPi AA-AMP+tRNA=>tRNA-AA+AMP

The enzyme binds adenosine triphosphate (ATP) and its specific aminoacid to catalyze formation of an aminoacyl adenylate complex (AA-AMP)with concomitant release of pyrophosphate (PPi). In the second step, theamino acid is transferred to the 2′ or 3′ terminus of the tRNA yielding“charged” tRNA and adenosine monophosphate (AMP). The charged tRNAdelivers the amino acid to the nascent polypeptide chain on theribosome.

There are at least twenty essential enzymes in this family for eachorganism. Inhibition of any of the essential tRNA synthetases disruptsprotein translation, ultimately resulting in growth inhibition.Pseudomonic acid A, an antibacterial agent currently used in humantherapy, provides clear evidence of the utility of tRNA synthetaseinhibitors as useful pharmaceuticals. Pseudomonic acid A binds to oneparticular tRNA synthetase, isoleucyl tRNA synthetase, and inhibitsisoleucyl adenylate formation in several Gram positive bacterialpathogens such as Staphylococcus aureus, resulting in the inhibition ofprotein synthesis, followed by growth inhibition. Novel syntheticcompounds that target tRNA synthetases offer clear advantages as usefultherapeutic agents to curb the threat of drug resistance. Drugresistance allows a pathogen to circumvent the biochemical disruptioncaused by an antimicrobial agent. This resistance can be a result of amutation that has been selected for and maintained. Pathogens in theenvironment have had repeated exposure to current therapeutics. Thisexposure has led to the selection of variant antimicrobial strainsresistant to these drugs.

Novel synthetic antimicrobial agents, therefore, would be expected to beuseful to treat drug resistant pathogens, since the pathogen has neverbeen exposed to the novel antimicrobial agent. The development ofcompounds or combinations of compounds targeting more than one tRNAsynthetase is also advantageous. Accordingly, inhibition of more thanone enzyme should reduce the incidence of resistance since multiplemutations in a pathogen would be required and are statistically rare.

SUMMARY OF THE INVENTION

The present invention discloses novel compounds which inhibit tRNAsynthetases and have efficacy, including whole cell killing, against abroad spectrum of bacteria and fungi. Described herein are compoundsthat exhibit tRNA synthetase inhibition.

The present invention comprises, in one aspect, compounds of Formula I.

Group Ar of Formula I is selected from aryl or heteroaryl. Preferably,Ar is aryl, more preferably, substituted phenyl, even more preferably,2,4-dichlorophenyl.

Group L of Formula I is selected from —C(O)N(Q)CH₂—, or—CR¹⁰R¹¹OCR¹²R¹³—; wherein Q is selected from hydrido, —(CH₂)_(m)CO₂H or—(CH₂)_(m)CO₂CH₃; and wherein m is a whole number from 1-4. Preferably,L is —C(O)NHCH₂—.

Each of substituents R¹, R², R⁹, R¹⁰, R¹¹, R¹², and R¹³ of Formula I isindependently selected from hydrido or lower alkyl, preferably hydrido.

Each of substituents R³, R⁴, R⁵, R⁶, R⁷, and R⁸ of Formula I isindependently selected from hydrido, acyl, amino, cyano, acyloxy,acylamino, carboalkoxy, carboxyamido, carboxy, halo, thio, alkyl,heteroaryl, heterocyclyl, alkoxy, aryloxy, sulfoxy,N-sulfonylcarboxyamido, N-acylamino sulfonyl, hydroxy, aryl, cycloalkyl,sulfinyl, or sulfonyl. Additionally, R³ and R⁴ together or R⁵ and R⁶together or R⁷ and R⁸ together are selected from

wherein each of R¹⁴, R¹⁵ and R¹⁶ is independently selected from hydrido,alkyl or carboxy-substituted alkyl; provided that at least five of R³,R⁴, R⁵, R⁶, R⁷, and R⁸ are independently hydrido. Preferably, each ofR³, R⁴, R⁵, R⁶, R⁷, and R⁸ is independently selected from hydrido,hydroxy, alkoxy, alkyl, amino, and carboxyamido. More preferably , eachof R³, R⁴, R⁵, R⁶, R⁷, and R⁸ is independently selected from hydrido,—O(CH₂)_(n)CO₂R¹⁷, —O(CH₂)_(n)CONHSO₂R¹⁸, —(CH₂)_(n)CO₂R¹⁹,—(CH₂)_(n)CONHSO₂R²⁰, —C(O)NHCH(R²²)CO₂R²¹, or —N(R²³)(CH₂)_(n)CO₂R²⁴,wherein each of R¹⁷, R¹⁹, R²¹, R²², R²³, and R²⁴ is independentlyselected from hydrido or alkyl; wherein each of R¹⁸ and R²⁰ isindependently alkyl; wherein n is selected from 1 or 2. Even morepreferably, each of R³, R⁴, R⁶, R⁷, and R⁸ is hydrido and R⁵ is selectedfrom —O(CH₂)_(n)CO₂R¹⁷, —O(CH₂),CONHSO₂R¹⁸, —(CH₂)_(n)CO₂R¹⁹,—(CH₂)_(n)CONHSO₂R²⁰, C(O)NHCH(R²²)—CO₂R²¹, or —N(R²³)(CH₂)_(n)CO₂R²⁴.

Group Het of Formula I is selected from

wherein X is selected from N or CR²⁷; wherein Y is selected from NH, Sor O; wherein Z is selected from N or CR²⁸; wherein each of R²⁵, R²⁶,R²⁷, and R²⁸ is independently selected from nitro, halo, hydroxy, loweramino, lower alkyl, lower alkoxy, aryloxy, lower carboalkoxy, sulfinyl,sulfonyl, carboxy, lower thio, and sulfoxy; and wherein each of R²⁹,R³⁰, and R³¹ is selected from hydrido, alkyl aryl, nitro, amino,sulfonyl or sulfinyl. Preferably, Het is

The invention also embraces pharmaceutically-acceptable salts of theforgoing compounds.

A further aspect of the invention comprises using a compositioncomprising the compound(s) of Formula I to inhibit a tRNA synthetase andin particular, to modulate the growth of bacterial or fungal organismsin mammals, a plant or a cell culture.

Yet another aspect of the invention involves a method of inhibiting thegrowth of microorganisms. The method involves exposing the microorganismto a compound of the invention, preferably a compound of Formula I,under conditions whereby a therapeutically effective amount of thecompound enters the microorganism. The method is useful for inhibitingthe growth of microrganisms in vivo and in vitro.

Another aspect of the invention is a pharmaceutical compositioncomprising the compound(s) of the invention and, in particular, thecompounds of Formula I, useful in the treatment of microbial infections,e.g., bacterial infections, fungal infections. A related aspect of theinvention is a method of making a medicament which involves placing acompound(s) of the invention, preferably a compound of Formula I, in asuitable pharmaceutically acceptable canner.

These and other aspects of the invention will be more apparent inreference to the following detailed description of the invention.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

Molecular terms, when used in this application, have their commonmeaning unless otherwise specified. The term “hydrido” denotes a singlehydrogen atom (H). The term “acyl” is defined as a carbonyl radicalattached to a hydrido, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycyl,aryl or heteroaryl group, examples of such radicals being formyl, acetyland benzoyl. The term “amino” denotes a nitrogen radical containing twosubstituents independently selected from the group consisting ofhydrido, alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl.Preferred amino radicals are NH₂ radicals and “lower amino” radicals,whereby the two substituents are independently selected from hydrido andlower alkyl. A subset of amino is “alkylamino”, whereby the nitrogenradical contains at least 1 alkyl substituent. Preferred alkylaminogroups contain alkyl groups that are substituted, for example, with acarboalkoxy group. The term “acyloxy” denotes an oxygen radical adjacentto an acyl group. The term “acylamino” denotes a nitrogen radicaladjacent to an acyl, carboalkoxy or carboxyamido group. The term“carboalkoxy” is defined as a carbonyl radical adjacent to an alkoxy oraryloxy group. The term “carboxyamido” denotes a carbonyl radicaladjacent to an amino group. A subset of carboxyamido is“N-sulfonylcarboxyamido” which denotes a carbonyl radical adjacent to anN-sulfonyl-substituted amino group. The term “halo” is defined as abromo, chloro, fluoro or iodo radical. The term “thio” denotes a sulfurradical adjacent to a substituent group selected from hydrido, alkyl,cycloalkyl, heterocyclyl, aryl and heteroaryl, such as, methylthio andphenylthio. Preferred thio radicals are “lower thio” radicals containinglower alkyl groups.

The term “alkyl” is defined as a linear or branched, saturated radicalhaving one to about ten carbon atoms unless otherwise specified.Preferred alkyl radicals are “lower alkyl” radicals having one to aboutfive carbon atoms. One or more hydrogen atoms can also be replaced by asubstitutent group selected from acyl, amino, acylamino, acyloxy,carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxy, nitro, thio,alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl,alkoxy, aryloxy, sulfoxy, sulfinyl, sulfonyl, N-sulfonylcarboxyamido,and N-acylaminosulfonyl. Preferred substituents are carboalkoxy,carboxy, N-sulfonylcarboxyamido, and N-acylaminosulfonyl. Examples ofalkyl groups include methyl, tert-butyl, isopropyl, methoxymethyl,carboxymethyl, and carbomethoxymethyl. The term “alkenyl” embraceslinear or branched radicals having two to about twenty carbon atoms,preferably three to about ten carbon atoms, and containing at least onecarbon-carbon double bond. One or more hydrogen atoms can also bereplaced by a substituent group selected from acyl, amino, acylamino,acyloxy, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxy,nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl,heteroaryl, alkoxy, aryloxy, sulfoxy, sulfinyl, sulfonyl,N-sulfonylcarboxyamido, and N-acylaminosulfonyl. Examples of alkenylgroups include ethylenyl or phenyl ethylenyl. The term “alkynyl” denoteslinear or branched radicals having from two to about ten carbon atoms,and containing at least one carbon-carbon triple bond. One or morehydrogen atoms can also be replaced by a substituent group selected fromacyl, amino, acylamino, acyloxy, carboalkoxy, carboxy, carboxyamido,cyano, halo, hydroxy, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl,heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfoxy, sulfinyl,sulfonyl, N-sulfonylcarboxyamido, and N-acylaminosulfonyl. Examples ofalkynyl groups include propynyl. The term “aryl” denotes aromaticradicals in a single or fused carbocyclic ring system, having from fiveto twelve ring members. One or more hydrogen atoms may also be replacedby a substituent group selected from acyl, amino, acylamino, acyloxy,carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxy, nitro, thio,alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl,alkoxy, aryloxy, sulfoxy, sulfinyl, sulfonyl, N-sulfonylcarboxyamido,and N-acylaminosulfonyl. Examples of aryl groups include phenyl,2,4-dichlorophenyl, naphthyl, biphenyl, terphenyl. “Heteroaryl” embracesaromatic radicals that contain one to four hetero atoms selected fromoxygen, nitrogen and sulfur in a single or fused heterocyclic ringsystem, having from five to fifteen ring members. One or more hydrogenatoms may also be replaced by a substituent group selected from acyl,amino, acylamino, acyloxy, carboalkoxy, carboxy, carboxyamido, cyano,halo, hydroxy, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl,heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfoxy, sulfinyl,sulfonyl, N-sulfonylcarboxyamido, and N-acylaminosulfonyl. Examples ofheteroaryl groups include, tetrazolyl, pyridinyl, thiazolyl,thiadiazoyl, isoquinolinyl, pyrazolyl, oxazolyl, oxadiazoyl, triazolyl,and pyrrolyl groups.

The term “cycloalkyl” is defined as a saturated or partially unsaturatedcarbocyclic ring in a single or fused carbocyclic ring system havingfrom three to twelve ring members. One or more hydrogen atoms may alsobe replaced by a substituent group selected from acyl, amino, acylamino,acyloxy, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxy,nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl,heteroaryl, alkoxy, aryloxy, sulfoxy, sulfinyl, sulfonyl,N-sulfonylcarboxyamido, and N-acylaminosulfonyl. Examples of acycloalkyl group include cyclopropyl, cyclobutyl, cyclohexyl, andcycloheptyl. The term “heterocyclyl” embraces a saturated or partiallyunsaturated ring containing zero to four hetero atoms selected fromoxygen, nitrogen and sulfur in a single or fused heterocyclic ringsystem having from three to twelve ring members. One or more hydrogenatoms may also be replaced by a substituent group selected from acyl,amino, acylamino, acyloxy, carboalkoxy, carboxy, carboxyamido, cyano,halo, hydroxy, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl,heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfoxy, sulfinyl,sulfonyl, N-sulfonylcarboxyamido, and N-acylaminosulfonyl. Examples of aheterocyclyl group include morpholinyl, piperidinyl, and pyrrolidinyl.The term “alkoxy” denotes oxy-containing radicals substituted with analkyl, cycloalkyl or heterocyclyl group. Examples include methoxy,tert-butoxy, benzyloxy and cyclohexyloxy. Preferred alkoxy radicals are“lower alkoxy” radicals having a lower alkyl substituent. The term“aryloxy” denotes oxy-containing radicals substituted with an aryl orheteroaryl group. Examples include phenoxy. The term “sulfinyl” isdefined as a tetravalent sulfur radical substituted with an oxosubstituent and a second substituent selected from the group consistingof alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl. The term“sulfonyl” is defined as a hexavalent sulfur radical substituted withtwo oxo substituents and a third substituent selected from alkyl,cycloalkyl, heterocyclyl, aryl and heteroaryl. The term“N-acylaminosulfonyl” denotes a hexavalent sulfur atom bound to two oxosubstituents and an N-acyl-substituted amino group.

The pharmaceutically-acceptable salts of the compounds of the invention(preferably a compound of Formula I) include acid addition salts andbase addition salts. The term “pharmaceutically-acceptable salts”embraces salts commonly used to form alkali metal salts and to formaddition salts of free acids or free bases. The nature of the salt isnot critical, provided that it is pharmaceutically-acceptable. Suitablepharmaceutically-acceptable acid addition salts of the compounds of theinvention (preferably a compound of Formula I) may be prepared from aninorganic acid or an organic acid. Examples of such inorganic acids arehydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric andphosphoric acid. Appropriate organic acids may be selected fromaliphatic, cycloaliphatic, aromatic, arylaliphatic, heterocyclic,carboxylic and sulfonic classes of organic acids, examples of which areformic, acetic, propionic, succinic, glycolic, gluconic, maleic, embonic(pamoic), methanesulfonic, ethanesulfonic, 2-hydroxyethanesulfonic,pantothenic, benzenesulfonic, toluenesulfonic, sulfanilic, mesylic,cyclohexylaminosulfonic, stearic, algenic, β-hydroxybutyric, malonic,galactic, and galacturonic acid. Suitable pharmaceutically-acceptablebase addition salts of compounds of the invention (preferably a compoundof Formula I) include, but are not limited to, metallic salts made fromaluminum, calcium, lithium, magnesium, potassium, sodium and zinc ororganic salts made from N,N′-dibenzylethylenediamine, chloroprocaine,choline, diethanolamine, ethylenediamine, N-methylglucamine andprocaine. All of these salts may be prepared by conventional means fromthe corresponding compound of the invention (preferably a compound ofFormula I) by treating, for example, the compound of the invention(preferably a compound of Formula I) with the appropriate acid or base.

As used herein, “treating” means preventing the onset of, slowing theprogression of, or eradicating the existence of the condition beingtreated, such as a microbial infection. Successful treatment ismanifested by a reduction and, preferably, an eradication of thebacterial and/or fungal infection in the subject being treated.

The compounds of the invention (preferably compounds of Formula I) canpossess one or more asymmetric carbon atoms and are thus capable ofexisting in the form of optical isomers as well as in the form ofracemic or non-racemic mixtures thereof. The compounds of the invention(preferably compounds of Formula I) can be utilized in the presentinvention as a single isomer or as a mixture of stereochemical isomericforms. Diastereoisomers can be separated by conventional means such aschromatography, distillation, crystallization or sublimation. Theoptical isomers can be obtained by resolution of the racemic mixturesaccording to conventional processes, for example by formation ofdiastereoisomeric salts by treatment with an optically active acid orbase. Examples of appropriate acids are tartaric, diacetyltartaric,dibenzoyltartaric, ditoluoyltartaric and camphorsulfonic acid. Themixture of diastereomers can be separated by crystallization followed byliberation of the optically active bases from these salts. Analternative process for separation of optical isomers includes the useof a chiral chromatography column optimally chosen to maximize theseparation of the enantiomers. Still another available method involvessynthesis of covalent diastereoisomeric molecules by reacting compoundsof the invention (preferably compounds of Formula I) with an opticallypure acid in an activated form or an optically pure isocyanate. Thesynthesized diastereoisomers can be separated by conventional means suchas chromatography, distillation, crystallization or sublimation, andthen hydrolyzed to obtain the enantiomerically pure compound. Theoptically active compounds of the invention (preferably compounds ofFormula I) can likewise be obtained by utilizing optically activestarting materials. These isomers may be in the form of a free acid, afree base, an ester or a salt.

The invention also embraces isolated compounds. An isolated compoundrefers to a compound which represents at least 10%, preferably 20%, morepreferably 50% and most preferably 80% of the compound present in themixture, and exhibits a detectable (i.e. statistically significant)antimicrobial activity when tested in conventional biological assayssuch as those described herein.

II. Description

According to one aspect of the invention, compounds of Formula I areprovided. The compounds are useful for inhibiting the enzymatic activityof a tRNA synthetase in vivo or in vitro. The compounds are particularlyuseful as antimicrobial agents, i. e., agents that inhibit the growth ofbacteria or fungi.

One sub-class of compounds of Formula I are compounds of Formula II

Substituents R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R²⁵, R²⁶, R²⁷ and R²⁸ are aspreviously described.

The compounds of the invention (preferably compounds of Formula I) areactive against a variety of bacterial organisms. They are active againstboth Gram positive and Gram negative aerobic and anaerobic bacteria,including Staphylococci, for example S. aureus; Enterococci, for exampleE. faecalis; Streptococci, for example S. pneumoniae; Haemophilus, forexample H. influenza; Moraxella, for example M. catarrhalis; andEscherichia, for example E. coli. The compounds of the present invention(preferably compounds of Formula I) are also active againstMycobacteria, for example M. tuberculosis. The compounds of the presentinvention (preferably compounds of Formula I are also active againstintercellular microbes, for example Chlamydia and Rickettsiae. Thecompounds of the present invention (preferably compounds of Formula I)are also active against Mycoplasma, for example M. pneumoniae.

The compounds of the present invention (preferably compounds of FormulaI) are also active against fungal organisms, including, among otherorganisms, the species Aspergillus, Blastomyces, Candida, Coccidioides,Cryptococcus, Epidermophyton, Hendersonula, Histoplasma, Microsporum,Paecilomyces, Paracoccidioides, Pneumocystis, Trichophyton, andTrichosporium.

In a second aspect the invention provides a pharmaceutical compositioncomprising a compound of the invention, preferably a compound inaccordance with the first aspect of the invention, and apharmaceutically-acceptable carrier (described below). As used hereinthe phrase “therapeutically-effective amount” means that amount of acompound of the present invention (preferably a compound of Formula I)which prevents the onset of, alleviates the symptoms of, or stops theprogression of a microbial infection. The term “microbial” meansbacterial and fungal, for example a “microbial infection” means abacterial or fungal infection. The term “treating” is defined asadministering, to a subject, a therapeutically-effective amount of acompound of the invention (preferably a compound of Formula I). The term“subject”, as described herein, is defined as a mammal, a plant or acell culture.

According to another aspect of the invention, a method for inhibiting atRNA synthetase is provided which comprises contacting a tRNA synthetasewith a compound of the invention (preferably a compound of Formula I)under the conditions whereby the tRNA synthetase interacts with itssubstrates and its substrates react(s) to form an aminoacyl adenylateintermediate and, preferably, react(s) further to form a charged tRNA.Such conditions are known to those skilled in the art (see also e.g.,the Examples for conditions), and PCT/US 96/11910, filed Jul.18, 1996(WO 97/05132, published Feb. 13, 1997), and U.S. Pat. No. 5,726,195.This method involves contacting a tRNA synthetase with an amount ofcompound of the invention (preferably a compound of Formula I) that issufficient to result in detectable tRNA synthetase inhibition. Thismethod can be performed on a tRNA synthetase that is contained within anorganism or outside an organism.

In a further aspect, the invention provides a method for inhibiting thegrowth of microorganisms, preferably bacteria or fungi, comprisingcontacting said organisms with a compound of the invention (preferably acompound of Formula I) under conditions which permit entry of thecompound into said organism and into said microorganism. Such conditionsare known to one skilled in the art and are exemplified in the Examples.This method involves contacting a microbial cell with atherapeutically-effective amount of compound(s) of the invention(preferably compound(s) of Formula I), e.g. to inhibit cellular tRNAsynthetase in vivo or in vitro. This method is used in vivo, forexample, for treating microbial infections in mammals. Alternatively,the method is used in vitro, for example, to eliminate microbialcontaminants in a cell culture, or in a plant.

In accordance with another aspect of the invention, the compositionsdisclosed herein are used for treating a subject afflicted by orsusceptible to a microbial infection. The method involves administeringto the subject a therapeutically effective amount of a compound of theinvention (preferably a compound of Formula I). According to this aspectof the invention, the novel compositions disclosed herein are placed ina pharmaceutically acceptable carrier and are delivered to a recipientsubject (preferably a human) in accordance with known methods of drugdelivery. Exemplary procedures for delivering an antibacterial,antifungal and antimycoplasmal agent are described in U.S. Pat. No.5,041,567, issued to Rogers and in PCT patent application numberEP94/02552 (publication no. WO 95/05384), the entire contents of whichdocuments are incorporated in their entirety herein by reference. Ingeneral, the methods of the invention for delivering the compositions ofthe invention ill vivo utilize art-recognized protocols for deliveringthe agent with the only substantial procedural modification being thesubstitution of the compounds of the invention (preferably compounds ofFormula I) for the drugs in the art-recognized protocols. Likewise, themethods for using the claimed composition for treating cells in culture,for example, to eliminate or reduce the level of bacterial contaminationof a cell culture, utilize art-recognized protocols for treating cellcultures with antibacterial agent(s) with the only substantialprocedural modification being the substitution of the compounds of theinvention (preferably compounds of Formula I) for the agents used in theart-recognized protocols.

The pharmaceutical preparations disclosed herein are prepared inaccordance with standard procedures and are administered at dosages thatare selected to reduce, prevent or eliminate the infection (See, e. g.,Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton,Pa. and Goodman and Gilman's The Pharmaceutical Basis of Therapeutics,Pergamon Press, New York, N.Y., the contents of which are incorporatedherein by reference, for a general description of the methods foradministering various antimicrobial agents for human therapy). Thecompositions of the invention (preferably of Formula I) can be deliveredusing controlled ( e.g., capsules) or sustained release delivery systems(e.g., bioerodable matrices). Exemplary delayed release delivery systemsfor drug delivery that are suitable for administration of thecompositions of the invention (preferably of Formula I) are described inU.S. Pat. Nos. 4,452,775 (issued to Kent), 5,239,660 (issued toLeonard), 3,854,480 (issued to Zaffaroni).

The pharmaceutically-acceptable compositions of the present inventioncomprise one or more compounds of the invention (preferably compounds ofFormula I) in association with one or more nontoxic,pharmaceutically-acceptable carriers and/or diluents and/or adjuvantsand/or excipients, collectively referred to herein as “carrier”materials, and if desired other active ingredients.

The compounds of the present invention (preferably compounds of FormulaI) are administered by any route, preferably in the form of apharmaceutical composition adapted to such a route, as illustrated belowand are dependent on the condition being treated. The compounds andcompositions can be, for example, administered orally, intravascularly,intraperitoneally, subcutaneously, intramuscularly or topically.

For oral administration, the pharmaceutical compositions are in the formof, for example, a tablet, capsule, suspension or liquid. Thepharmaceutical composition is preferably made in the form of a dosageunit containing a therapeutically-effective amount of the activeingredient. Examples of such dosage units are tablets and capsules. Fortherapeutic purposes, the tablets and capsules which can contain, inaddition to the active ingredient, conventional carriers such as bindingagents, for example, acacia gum, gelatin, polyvinylpyrrolidone,sorbitol, or tragacanth; fillers, for example, calcium phosphate,glycine, lactose, maize-starch, sorbitol, or sucrose; lubricants, forexample, magnesium stearate, polyethylene glycol, silica, or talc;disintegrants, for example, potato starch, flavoring or coloring agents,or acceptable wetting agents. Oral liquid preparations generally are inthe form of aqueous or oily solutions, suspensions, emulsions, syrups orelixirs may contain conventional additives such as suspending agents,emulsifying agents, non-aqueous agents, preservatives, coloring agentsand flavoring agents. Examples of additives for liquid preparationsinclude acacia, almond oil, ethyl alcohol, fractionated coconut oil,gelatin, glucose syrup, glycerin, hydrogenated edible fats, lecithin,methyl cellulose, methyl or propyl para-hydroxybenzoate, propyleneglycol, sorbitol, or sorbic acid.

The pharmaceutical compositions can be administered via injection.Formulations for parenteral administration can be in the form of aqueousor non-aqueous isotonic sterile injection solutions or suspensions.These solutions or suspensions can be prepared from sterile powders orgranules having one or more of the carriers mentioned for use in theformulations for oral administration. The compounds can be dissolved inpolyethylene glycol, propylene glycol, ethanol, corn oil, benzylalcohol, sodium chloride, and/or various buffers.

For topical use the compounds of the present invention can also beprepared in suitable forms to be applied to the skin, or mucus membranesof the nose and throat, and can take the form of creams, ointments,liquid sprays or inhalants, lozenges, or throat paints. Such topicalformulations further can include chemical compounds such asdimethylsulfoxide (DMSO) to facilitate surface penetration of the activeingredient.

For application to the eyes or ears, the compounds of the presentinvention can be presented in liquid or semi-liquid form formulated inhydrophobic or hydrophilic bases as ointments, creams, lotions, paintsor powders.

For rectal administration the compounds of the present invention can beadministered in the form of suppositories admixed with conventionalcarriers such as cocoa butter, wax or other glyceride.

Alternatively, the compounds of the present invention can be in powderform for reconstitution in the appropriate pharmaceutically acceptablecarrier at the time of delivery.

The dosage regimen for treating an infection with the compound and/orcompositions of this invention is selected in accordance with a varietyof factors, including the type, age, weight, sex and medical conditionof the patient, the severity of the infection, the route and frequencyof administration and the particular compound employed. In general,dosages are determined in accordance with standard practice foroptimizing the correct dosage for treating an infection.

The compositions can contain from 0.1% to 99% by weight, preferably10-60% by weight, of the active ingredient, depending on the method ofadministration. If the compositions contain dosage units, each dosageunit preferably contains from 50-500 mg of the active material. Foradult human treatment, the dosage employed preferably ranges from 100 mgto 3 g, per day, depending on the route and frequency of administration.

If administered as part of a total dietary intake, the amount ofcompound employed can be less than 1% by weight of the diet andpreferably no more than 0.5% by weight. The diet for animals can benormal foodstuffs to which the compound can be added or it can be addedto a premix.

Further references to features and aspects of the invention are providedin the Examples set out hereafter.

EXAMPLES

The following Examples are detailed descriptions of the methods ofpreparation of compounds of Formula I. These detailed preparations fallwithin the scope of, and serve to exemplify, the invention. TheseExamples are presented for illustrative purposes only and are notintended as a limitation on the scope of the invention.

Synthesis of I

To 10.0 g of 4-hydroxy-L-proline methylester in 100 ml of anhydroustethydrofuran was added 10.5 ml of 2,4-dichlorobenzylchloride, 30 ml ofdiisopropylethylamine, and 100 mg of tetrabutylammonium iodide,respectively. The reaction was allowed to stir for 16 hours at roomtemperature before partitioning with 200 ml ethyl acetate and 300 ml 1 Nhydrochloric acid. The acid layer was neutralized with sodium hydrogencarbonate and extracted with 300 ml ethyl acetate. The organic layer wasdried with 10 g sodium sulfate and poured through 100 g of silica gel.The solution was concentrated to yield 16.8 g of I as a clear oil.

Synthesis II

To 16.8 g of I in 50 ml anhydrous N,N′-dimethylformamide was added 9.2 gof tert-butyldimethylsilyl chloride followed by 4.5 g imidazole. Thereaction was allowed to stir at room temperature for 16 hours beforepartitioning with 300 ml ethylacetate and (2×400 ml) brine. The organiclayer was dried with 10 g sodium sulfate and poured through 100 g ofsilica gel. The solution was concentrated to afford 23 g of II 1as ayellow oil.

Synthesis III

At 0° C., 23.0 g of II in 50 ml methanol and 50 ml 1,4-dioxane was addedto a solution of 2.5 g lithiumhydroxide monohydrate in 25 ml water.After 1 hour, the reaction mixture was partitioned with 250 ml ethylacetate and 250 ml dilute citric acid. The organic layer was washed with200 ml brine then dried with 10 g sodium sulfate. Concentration in vacuyielded 19.1 g of II as a yellow oil.

Synthesis IV

To 0.36 g III in 10 ml anhydrous N,N′-dimethylformamide was added 0.26 g2-(aminomethyl)benzimidazole dihydrochloride, 1.1 mldiisopropylethylamine and 0.22 g1-(3-dimethylaminoproply)-3-ethylcarbodiimide hydrochloride,respectively. The reaction was stirred for 16 hours at room temperaturebefore partitioning with 30 ml ethylacetate and 2×50 ml brine. Theorganic layer was dried with 0.5 g sodium sulfate then concentrated todryness. Purification by silica gel chromatography gave 0.20 g of IV.

Synthesis V

A solution of 0.20 g IV in 4 ml of 1 M tetrabutylammonium fluoride intetrahydrofuran was stirred at room temperature for 16 hours. Thereaction was concentrated and purified by silica gel chromatographyusing 10% methanol in dichloromethane to give 0.07 g of V as a whitesolid.

Synthesis of VI

A solution of 5.0 g of S-pyrrolidine methanol, 7.6 ml of 2,4-dichlorobenzyl chloride, 17.3 ml diisopropylethylamine and 0.1 gtetrabutylammoniam iodide in 100 ml anhydrous tetrahydrofuran wasstirred at room temperature for 18 hours before partitioning with 200 mlethylacetate and 200 ml 1N hydrochloric acid. The acid layer wasneutralized with sodium bicarbonate then extracted with 200 ml ethylacetate. The organic layer was washed with 200 ml brine and dried with10 g sodium sulfate. Concentration of the organic solution gave 10.3 gof VI as an oil.

Synthesis of VII

0.43 g of VI was added to 0.07 g of 60% NaH in 10 ml anhydrousN,N′-Dimethylformamide. After stirring at room temperature for 1 hour,the chlormethylbenzimidazole II was added. The reaction was stirred for18 hours before partitioning with 50 ml ethyl acetate and 50 ml brine.The organic layer was dried with 5 g sodium sulfate and concentrated.The crude oil was purified by silica gel chromatography using 1:1hexane/ethyl acetate to give 0.56 g of VII.

Synthesis of VIII

To 0.56 g of VII in 5 ml 1,4-dioxane was added 0.2 ml concentratedhydrochloric acid. The reaction was heated at 100° C. for 2 hours beforepartitioning with 30 ml ethyl acetate and 30 ml saturated solution ofsodium bicarbonate. The organic layer was washed with 30 ml brine anddried with 2 g sodium sulfate. Concentration of the organic layerafforded 0.4 g of VIII as an oil.

Synthesis of N-alkylated hydroxy proline methyl esters (II)

To a suspension of hydroxy-L-Proline methyl ester hydrochloride (I,1.1mmol, 200 mg) in 3 ml dichloromethane in a 16 mm tube with a screw caplid was added diisopropylethylamine (DIEA, 2.43 mmol, 0.43 ml). Thesuspension was sonicated for 2 minutes, then aryl chloride (R₁CH₂Cl,1.05 mmol) and tetrabutylammonium iodide (TBAI, 0.05 mmol, 20 mg) wereadded. Reaction mixture was heated at 40° C. for 24 hr then cooled toroom temperature and diluted with 5 ml of dichloromethane and 5 ml ofaqueous saturated sodium bicarbonate. The reaction mixture was shakenvigorously and the layers were separated. The organic layer wascollected in a clean 16 mm tube and the solvent was evaporated undernitrogen stream to yield crude N-alkylated hydroxy proline II. Eachintermediate was characterized by LC/MS and yielded a major peakcorresponding to the molecular ion.

Trans-3-hydroxy-L-proline methyl ester hydrochloride (I, X═Z═H, Y═OH)was prepared by dissolving the corresponding trans-3-hydroxy-L -proline(7.6 mmol, 1 g) in 20 ml of methanol and 15 ml of 1M hydrochloric acidin ether. The reaction mixture was refluxed for 3 hr, then cooled theroom temperature and stripped in vacuo to yield a white solid (1.2 g).

Aryl chlorides used for these displacements were obtained fromcommercial sources: 2-chlorobenzyl chloride (0.14 ml),2,6-dichlorobenzyl chloride (215 mg), 6-chloropiperonyl chloride (225mg), 2-chloro-4-nitrobenzyl chloride (226 mg), 3,4-dichlorobenzylchloride (0.15 ml), 2,3-dichlorobenzyl chloride (0.15 ml),2,5-dichlorobenzyl chloride (0.15 ml), 2,4-dichlorobenzyl chloride (0.15ml), and 2-(4-chlorophenyl)-4-chloromethyl thiazole (265 mg).

Hydrolysis of methyl esters to acids (III)

To a glass tube containing a suspension of II (1.05 mmol) in 6 ml of 1:1mixture of THF and water was added lithium hydroxyde monohydrate (2.2mmol, 53 mg). The reaction mixture was stirred at room temperature for 2hrs to yield a clear, colorless solution that was then diluted with 10ml of saturated aqueous sodium bicarbonate. The aqueous layer was washedthree times with 5 ml of dichloromethane and the organic phase wasdiscarded. The aqueous phase was then acidified to pH 1 with 6Nhydrochloric acid, frozen and lyophilized overnight. The acid (III) wasanalyzed by LC/MS and yielded pure material corresponding to themolecular ion.

Acid intermediate in the synthesis of amide compound 8 (Table 1a): δ_(H)(DMSO-d6) 1.65 (1H, m), 2.05 (2H, m), 3.10 (2H, m), 3.30 (1H, m), 3.55(1H, d, J=15 Hz), 4.10 (1H, d, J=15 Hz), 7.36 (1H, dd, J₁=2.0 Hz, J₂=8.5Hz), 7.49 (1H, d, J=2.0 Hz), 7.62 (1H, d, 8.5 Hz).

Amide formation (IV)

Acid (III, 0.18 mmol) was dissolved in 0.8 ml of anhydrous acetonitrileand 0.2 ml of DIEA. To this slightly cloudy solution were added amine(0.18 mmol), hydroxybenzotriazole hydrate (0.18 mmol) and finally1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). The reactionmixtures were stirred at room temperature for 16 hours, then dilutedwith 2 ml of ethyl acetate. The organic layer was washed with 3 ml ofsaturated aqueous sodium bicarbonate and collected into a 16 mm testtube. The solvent was evaporated under a nitrogen stream and solidmaterial was redissolved in 2 ml of 1:1 water/acetonitrile mixture.Sample was purified by HPLC using YMC-Pack ODS (100×20 mm) column;eluting with a gradient of 90% 0.1% TFA/water to 100% 0.1% TFA inacetonitrile over 10 min. at 20 ml/min, detecting at 254 nm. The largestpeak was collected and analyzed by LC/MS (ESI) to yield a single UV peakcorresponding to the molecular ion. Pure fully elaborated product wasobtained as a bis-TFA salt by freezing, and lyophilizing the fractioncontaining the largest peak.

Compound 8, (Table 1a): δ_(H) (CD₃OD) 2.23 (1H, m), 2.58 (1H, m), 3.36(1H, m), 3.76 (1H, dd, J₁=4.5 Hz, J₂=12.5 Hz), 4.58 (2H, m), 4.63 (1H,m), 4.70 (1H, d, J=13.2 Hz), 4.78 (1H, d, J=16.4 Hz), 4.88 (1H, d,J=16.4 Hz), 7.32 (1H, dd, J₁=2.0 Hz, J₂=8.4 Hz), 7.48 (1H, d, J=2.0),7.58 (3H, m), 7.78 (2H, m).

Amines used in this sequence were: 2-(aminomethyl)benzimidazoledihydrochloride, 2-(aminomethyl )-4,5-dimethylbenzimidazole,2-(aminomethyl)-4-carboxymethylbenzimidazole,2-(aminomethyl)-4-chlorobenzimidazole,2-(aminomethyl)-4,5-dichlorobenzimidazole,2-(aminomethyl)-4-aminobenzimidazole. These benzimidazole analogs wereprepared according to the procedure described by Keenan, R. M. et al.(Bioorg. Med. Chem Lett. 1998, 8, 3165-3170).

Herein below, Tables 1a and 1b provide 4-hydroxyproline derivatives withunsubstituted and substituted benzimidazoles, respectively; Table 2aprovides 3-hydroxyproline analogs with unsubstituted benzimidazoles; andTable 2b provides 3-hydroxyproline analogs with substitutedbenzimidazoles.

TABLE 1a 4-hydroxyproline derivatives containing unsubstitutedbenzimidazole

Cmpd. X Z R1 Structure MS Data 1 OH H

385(M + H⁺) 2 OH H

419(M + H⁺) 3 OH H

429(M + H⁺) 4 OH H

430(M + H⁺) 5 OH H

419(M + H⁺) 6 OH H

419(M + H⁺) 7 OH H

419(M + H⁺) 8 OH H

419(M + H⁺) 9 OH H

468(M + H⁺) 10 H OH

419(M + H⁺) 11 H H

403(M + H⁺)

TABLE 1b trans-4-hydroxyproline derivatives containing substitutedbenzimidazoles

Cmpd R1 R2 Structure MS Data 12

447 (M + H⁺) 13

477 (M + H⁺) 14

453 (M + H⁺) 15

487 (M + H⁺)

TABLE 2a trans-3-hydroxyproline derivatives containing unsubstitutedbenzimidazoles

Cmpd R1 Structure MS Data 16

419(M + H⁺) 17

429(M + H⁺) 18

468(M + H⁺) 19

419(M + H⁺)

TABLE 2b trans-3-hydroxyproline derivatives containing substitutedbenzimidazoles

Cmpd. R1 R2 Structure MS Data 20

447(M + H⁺) 21

477(M + H⁺) 22

453(M + H⁺) 23

453(M + H⁺) 24

487(M + H⁺) 25

496(M + H⁺) 26

426(M + H⁺) 27

502(M + H⁺) 28

457(M + H⁺) 29

487(M + H⁺) 30

463(M + H⁺)

To a solution of trans-4-hydroxy-L-proline methyl ester hydrochloride(I,2.07 mmol, 300 mg) and aldehyde (R₁CHO, 2.07 mmol) in 7 ml ofdichloethylene were added triethylamine (4.14 mmol, 0.58 ml) andsodiumtriacetoxyborohydride (2.9 mmol, 613 mg). The cloudy reactionmixture was stirred at room temperature for 3 hr then quenched withsaturated aqueous sodium bicarbonate (6 ml). The aqueous layer wasextracted three times with 7 ml of ethyl acetate. The organic layerswere combined, dried over anhydrous sodium sulfate and stripped in vacuoto yield a product requiring no further purification, as judged by itsLC/MS trace.

The alkylated proline methyl ester (II) was elaborated to final amide(IV) using the procedure described in Scheme III.

Table 3, below, provides examples of alkylated prolines obtained throughreductive amination.

TABLE 3 trans-4-hydroxyproline derivatives synthesized using reductiveamination

Cmpd. R1 Structure MS Data 31

429(M + H⁺) 32

441(M + H⁺) 33

447(M + H⁺) 34

435(M + H⁺) 35

473(M + H⁺) 36

453(M + H⁺) 37

419(M + H⁺) 38

435(M + H⁺) 39

485(M + H⁺)

To a solution of methyl ester (II, 0.1 mmol, 30 mg) in 1 ml of anhydrousDMF were added sequentially sodium hydride (60 mol %, 0.11 mmol, 3 mg)and alkyl bromide. The reaction was stirred at room temperature for 16hr. Crude reaction mixture was analyzed by LC/MS and showed alkylatedproduct as the major UV component of the trace. The reaction wasquenched with 2 ml of 1:1 THF/H₂O mixture, then lithium hydroxidemonohydrate was added (0.2 mmol 5 mg) to the reaction. After 2 hr,reaction mixture was worked up as described in Scheme III (hydrolysis ofmethyl esters). The final amide products were prepared as described inScheme III.

Compounds 44 and 47 of Table 4 were prepared by cleavage of thecorresponding tert-butyl esters, compounds 43 and 46, respectively. Thecleavage was carried out in 1 ml of 40% trifluoroacetic acid indichloromethane over 1 hr. The final products were isolated byevaporation of solvent in vacuo and characterized by LC/MS.

Table 4, below, provides examples of 3- and 4-alkoxyproline derivatives.

TABLE 4 3- and 4-alkoxyproline derivative

Cmpd. X Y Structure MS Data 40 H

457(M + H⁺) 41 H

509(M + H⁺) 42 H

459(M + H⁺) 43 H

533(M + H⁺) 44 H

477(M + H⁺) 45

H

457(M + H⁺) 46

H

533(M +H⁺) 47

H

477(M + H⁺) 48

H

528(M + H⁺)

Proline analogue I (Compound 10, Table 1a, 0.17 mmol, 72 mg) wasdissolved in 3 ml of dichloromethane and cooled to 0° C. The reactionmixture was treated with anhydrous pyridine (0.7 mmol, 0.06 ml) andmethanesulfonyl chloride (0.38 mmol, 0.03 ml). Reaction mixture wasslowly warmed up to room temperature over 10 hr then quenched with 5 mlof aqueous saturated sodium bicarbonate. The layers were separated andthe aqueous layer washed two more times with 5 ml dichloromethane. Theorganic layers were combined, dried over anhydrous sodium sulfate andstripped to yield 80 mg of yellowish solid (II) which was characterizedby LC/MS (575 M+H⁺) and required no further purification.

To bismesylate II (20 mg, 0.035 mmol), dissolved in 0.5 ml anhydrousdimethylformamide was added solid sodium hydride (60%, 0.18 mmol, 7 mg).Reaction mixture was placed under nitrogen atmosphere, thiol (0.18 mmol)was added and mixture was heated at 40 C. for 16 h. Reaction wasquenched with 2 ml water and washed two times with 3 ml dichloromethane.Organic layers were combined, dried over anhydrous sodium sulfate andevaporated to dryness under stream of nitrogen. Crude products werepurified by preparative HPLC as described in Scheme III and isolated asbis TFA salts upon lyophilization.

Compound 49 (Table 5): δ_(H)(CDCl₃): 1.30 (5H, m), 1.62 (1H, m), 1.75(2H, m), 1.93 (2H, m), 2.58 (2H, m), 2.75 (1H, m), 2.92 (1H, m), 3.60(1H, m), 3.95 (1H, m), 4.35 (2H, m), 4.54 (1H, m) 5.04 (2H, m), 7.16(1H, dd, J=8.3 Hz, J=2.0 Hz), 7.48 (2H, m), 7.54 (1H, d, J=8.3 Hz), 7.71(2H, m).

Compounds 52 and 53 (acids) were prepared by hydrolysis of thecorresponding Compounds 51 and 54 (esters), respectively, as describedin Scheme III (hydrolysis of methyl esters to acids). The acids wereisolated by preparative HPLC followed by lyophilization.

Table 5, below, provides examples of 4-thioether proline derivatives.

TABLE 5 4-thioether proline derivatives

Cmpd. R₁ Structure MS Data 49

517(M + H⁺) 50

479(M + H⁺) 51

521(M + H⁺) 52

507(M + H⁺) 53

522(M + H⁺) 54

550(M + H⁺)

2-Aminomethylimidazopyridine (2a, b) and 2-aminomethylimidazopyrimidines(2c, d) were prepared by modification of procedure reported by S. Takadaet al. (J. Med. Chem. 1996, 39, 2844-2851). Sample procedure belowdescribes preparation of 3b. The same method was carried out inpreparation of 3a, 3c, and 3d.

A solution of N-Cbz-Glycine (1.5 mmol, 316 mg) in 3 ml of 10:1 mixtureof hexamethylphosphoramide (HMPA) and acetonitrile was placed undernitrogen atmosphere and cooled to 0° C. Thionyl chloride was addeddrop-wise with a syringe over 3 minutes and the solution was stirred at0° C. After 1 hr, 3,4-diaminopyridine (1b) was added (1.37 mmol, 150mg). The solution was left in an ice bath for 4 hr, then poured into 50ml ice-water and neutralized with saturated aqueous sodium bicarbonate.Aqueous layer was washed 4 times with 60 ml of ethyl acetate. Theorganic washes were combined, dried over anhydrous sodium sulfate andstripped to yield 2.5 ml of yellowish liquid MS (ESI): M+H⁺=301.

The solution was diluted with 3 ml HMPA and 2 ml glacial acetic acid andheated to 180° C. After 1.5 hr, the brown reaction mixture was cooled toroom temperature and poured into 70 ml saturated aqueous sodiumbicarbonate solution. The aqueous layer was washed 4 times with 100 mlportions of ethyl acetate. The organic washes were combined, dried overanhydrous sodium sulfate and stripped to yield a brown liquid that wasdivided into 3 portions and each portion dissolved in 2 ml methylenechloride. Each solution was then loaded onto a 2 g strong cationexchange cartridge (Varian Mega Bond Elut SCX) and washed with 5 mlmethylene chloride, 10 ml methanol, and finally with 5 ml 2Mammonia/methanol which was collected into a 25 ml round-bottomed flask.The solvent was removed in vacuo to yield 310 mg of 2b; δ_(H) (CDCl₃):4.45 (2H, br. s), 4.98 (2 H, s), 7.20 (5H, m) 7.45 (1H, d, J=6 Hz), 8.18(1H, d, J=6 Hz), 8.71 (1H, br. s); MS (ESI) M+H⁺283.

To a solution of 2b (70 mg, 0.25 mmol) in 8 ml of methanol was added 70mg of 10% Pd/C Degussa type (50% water content). The solution was placedunder hydrogen atmosphere and left to stir vigorously at roomtemperature. After 24 hr, the catalyst was filtered and washed with 50ml methanol and 3 ml DMF. The filtrates were collected and stripped toyield 35 mg of oily product. The amide 3b was prepared following theamide preparation procedure described for Scheme III.

Table 6, below, provides imidazopyridine and imidazopyrimidinederivatives of proline.

TABLE 6 Imidazopyridine and Imidazopyrimidine Derivatives of Proline

Cmpd. R₂ Structure MS Data 55

404(M + H⁺) 56

404(M + H⁺) 57

405(M + H⁺) 58

420(M + H⁺)

Biological Evaluation

Enzymatic Activity

IC₅₀ determinations for the aminoacyl-tRNA synthetases (aaRS) isolatedfrom pathogen or HeLa cells were carried out using a modification of theaaRS charging and trichloroacetic acid precipitation assay describedpreviously (see examples: D. Kern et. al., Biochemie, 61, 1257-1272(1979) and J. Gilbart et. al. Antimicrobial Agents and Chemotherapy,37(1), 32-38 (1993)). The aaRS enzymes were prepared via standardcloning and expression methods and partially purified or partiallypurified from pathogen and HeLa cell extracts. The activity of each aaRSenzyme was standardized as trichloroacetic acid precipitable counts(cpm) obtained at 10 minutes reaction time at K_(m) concentrations ofsubstrates. For practical purposes, the minimal acceptable value isapproximately 2000 cpm per 10 minute reaction.

Preincubations for IC₅₀ determinations were initiated by incubatingpartially purified aaRS extracts in 50 mM HEPES (pH 7.5), 0.1 mM EDTA,0.05 mg/ml bovine serum albumin, 10 mM dithiothreitol and 2.5% dimethylsulfoxide with and without test compound (e.g. compound of the invention(preferably a compound of Formula I)) in a final volume of 20microliters in a microtiter plate for 20 minutes at 25 C. Test compoundswere typically present as serial dilutions in concentration ranges of0.35 nM to 35 μM. Test compound solutions were prepared by dissolvingtest compound in 100% dimethyl sulfoxide and diluting to the finalconcentration with 50 mM HEPES, pH 7.5. IC₅₀ determinations weretypically performed in duplicate with each experiment containing 4-8concentrations of inhibitor along with two no inhibitor controls.

IC₅₀ incubations were initiated by supplementing the preincubationmixture to a final assay concentration of 10 mM MgCl₂, 30 mM KCl, 10 mMKF, 50 mM HEPES (pH 7.5), 20 μM-500 mM ATP, 2-20 μM [³H] amino acid, and90-180 μM crude tRNA in a final volume of 35 microliters. The reactionwas incubated at 25° C. for 5-20 minutes. At specified time points a 15microliter aliquot was removed and added to a well of a Milliporefiltration plate (Multiscreen-FB, MAFB NOB 10) containing 100microliters of cold 5% (wt/vol) trichloroacetic acid. Trichloroaceticacid precipitable material was collected by filtration on MilliporeMultiscreen filtration station, washed twice with cold 5%trichloroacetic acid, twice with water, and dried. Plates were typicallyallowed to air dry for several hours or they were baked at 50° C. in avacuum oven for 30 minutes. The radioactivity on the dried plates wasquantitated by the addition of Packard Microscint-20 to the wells andcounting with a Packard TopCount scintillation counter.

Inhibitor activity was typically reported as a percentage of the controlaaRS activity. The IC₅₀ value was determined by plotting per centactivity versus compound concentration in the assay and identifying theconcentration at which 50% of the activity was remaining.

The IC₅₀ values (in μM) of representative compounds of the presentinvention are listed in Table 8.

Whole Cell Antimicrobial Screens

Compounds were tested for antimicrobial activity against a panel oforganisms according to standard procedures described by the NationalCommittee for Clinical Laboratory Standards (NCCLS document M7-A3, Vol.13, No. 25, 1993/NCCLS document M27-P, Vol. 12, No. 25, 1992). Compoundswere dissolved in 100% DMSO and were diluted to the final reactionconcentration (0.1 μg/ml-500 μg/ml) in microbial growth media. In allcases the final concentration of DMSO incubated with cells is less thanor equal to 1%. For minimum inhibitory concentration (MIC) calculations,2-fold dilutions of compounds were added to wells of a microtiter platecontaining 1×10⁵ bacteria or fungal cells in a final volume of 200lambda of an appropriate media (Mueller-Hinton Broth; Haemophilus TestMedia; Mueller-Hinton Broth+5% Sheep Blood; or RPMI 1690). Plates wereincubated overnight at an appropriate temperature (30° C.-37° C.) andoptical densities (measure of cell growth) were measured using acommercial plate reader. The MIC value is defined as the lowest compoundconcentration inhibiting growth of the test organism. The MIC values (inμg/ml) of representative compounds of the present invention are listedin Table 8.

TABLE 8 Biological Activity

IC50 (nM)* MIC (μg/mL) CB # L R₅ n MS calcd MS obsv +/− ion Sa Ef Sa EfsEfm 126,881 (S)-CONHCH₂ H 1 403.1092 403.1109 + <500  <10000   100 <100127,006 (S)-CONHCH₂ (R,S) OH 1 419.1041 419.1030 + <500  <10000 <100<100 <100 127,566 (S)-CONHCH₂ (R,S)-CN 1 428    428    + <10000<10000 >100 >100 >100 127,889 (S)-CONHCH₂ (R,S)-OCH₂Ph 1 509.1511509.1532 + <10000 <10000 >100 >100 >100 130,692 (S)-CONHCH₂ ═O 1417.0885 417.0867 + <10000 <10000 >100 >100 >100 130,693 (S)-CONHCH₂ H 2417.1249 417.1232 + <10000 <10000 <100 >100   100 130,705 (S)-CONHCH₂═NNH₂ 1 421.1154 421.1151 + <10000 <10000 >100 >100 >100 130,706(S)-CONHCH₂ (R,S)-tetrazole 1 471.1215 471.1228 + <500 <500  >100 >100 >100 130,707 (S)-CONHCH₂ ═NOH 1 432.0994 432.0985 +<10000 <10000 >100 >100 >100 130,708 (S)-CONHCH₂ ═NOCH3 1 446.1150446.1170 + <10000 <10000 >100 >100 >100 130,709 (S)-CONHCH₂ ═NOCH₂CO₂H 1490.1049 490.1027 + <10000 <10000 >100 >100 >100 130,724 (R)-CONHCH₂ H 1403.1092 403.1104 + <10000 <10000 <100 >100 >100 130,725 (R)-CONHCH₂(R)-OH 1 419.1041 419.1029 + <60000 <60000 >100 >100 >100 130,900(S)-CONHCH₂ (S)-OH 1 419.1041 419.1061 + <500  <500    100 <100   100130,901 (S)-CONHCH₂ (R)-OH 1 419.1041 419.1038 + <500  <1000  <100 <100<100 130,928 (S)-CH₂OCH₂ H 1 390.1140 390.1138 + <10000 <10000  100 >100   100 130,973 (S)-CONHCH₂ CH₂CO₂H 1 461.1147 461.1126 + <500 <500  >100 >100 >100 *<500 = 500 nM or less; <1000 = 501-1000 nM; <10000= 1001-10000 nM; <60000 = 10001-60000 nM Sa = S. aureus Ef = E. faecalisEfm = E. faecium

In Vivo Efficacy

Mouse Protection Test

The mouse protection test is an industry standard for measuring theefficacy of a test compound in vivo [for examples of this model see J.J. Clement, et al., Antimicrobial Agents and Chemotherapy, 38 (5),1071-1078, (1994)]. As exemplified below, this test is used to show thein vivo efficacy of the compounds of the present invention againstbacteria or fungi.

The in vivo antimicrobial activity of a compound of the invention(preferably a compound of Formula I) hereinafter referred to as testcompound, is established by infecting male or female mice (5 mice/dosegroup×5 doses/compound) weighing 20-25 g intraperitoneally with pathogeninoculum. The inoculum is prepared from a sample of pathogen obtainedfrom the ATCC (for example, ATCC 29213, S. aureus; ATCC 14154, S.aureus;ATCC 8668, Strep. pyogenes; ATCC 25922, E. coli; ATCC 29212, E.faecalis; ATCC 25238, M. catarrhalis; and ATCC 90028, C. albicans). Eachbacterial strain is grown in its appropriate medium at 37° C. for 18 hr,most strains yielding between 10⁸ and 10⁹ colony forming units (CFU)/mlunder these conditions. The overnight culture is serially diluted to anappropriate content and then 0.5 ml of each dilution is added to 4.5 mlof 5% hog gastric mucin to prepare the infecting inoculum. Each mouse isinjected with 0.5 ml of the inoculum intraperitoneally (i.p.), fiveanimals per dilution. The 50% lethal dose (LD₅₀) and the minimal lethaldose (MLD, the dose causing 100% death of the animals) is calculated onthe basis of the number of mice surviving after 7 days. The MLDestablished for each of the pathogens is used as inoculum dose in themouse protection tests.

The test compound is dissolved in a sterile vehicle appropriate for itsmethod of delivery (for example, 30% HPB (hydroxypropyl-β-cyclodextrin),pH, 7.4 or 0.05M Tris.HCl). A vehicle group (dose=0) serves as a placebocontrol for each compound and each pathogen. The dose for the testcompound is determined based on the MIC data. A series of dilutions of atest compound is prepared in the vehicle. A group of 5 mice are used foreach test compound dose and the vehicle. There are 5-6 doses for eachcompound. Each animal is used for one experiment only.

Mice are infected i.p. with 0.5 ml of the MLD of pathogen in 5% hoggastric mucin by one researcher and immediately administered compound(s.c., p.o. or i.v. in volumes indicated above) by a second researcher.The 50% protective dose (PD₅₀) is calculated from the dose responsecurve established on the basis of the numbers of mice surviving for 7days after treatment. In each experiment, a group of positive controlwith a commercially available antibiotic for example, is also included.

All of the references, patents and patent publications identified orcited herein are incorporated, in their entirety, by reference.

Although this invention has been described with respect to specificembodiments, the details of these embodiments are not to be construed aslimitations. Various equivalents, changes and modifications may be madewithout departing from the spirit and scope of this invention, and it isunderstood that such equivalent embodiments are part of this invention.

What is claimed:
 1. A compound of the general formula:

(a) wherein Ar is selected from the group consisting of substituted orunsubstituted aryl, and substituted or unsubstituted heteroaryl; (b)wherein L is selected from the group consisting of —C(O)N(Q)CH₂—, and—CR¹⁰R¹¹OCR¹²R¹³—; wherein Q is selected from the group consisting ofhydrido, —(CH₂)_(m)CO₂H and —(CH₂)_(m)CO₂CH₃; wherein m is selected fromthe group consisting of 1, 2, 3, and 4; (c) wherein each of R¹, R², R⁹,R¹⁰, R¹¹, R¹² and R¹³ is independently selected from the groupconsisting of hydrido and lower alkyl; (d) wherein each of R³, R⁴, R⁵,R⁶, R⁷, and R⁸ is independently selected from the group consisting ofhydrido, acyl, amino, cyano, acyloxy, acylamino, carboalkoxy,carboxyamido, carboxy, halo, alkyl, heteroaryl, heterocyclyl, alkoxy,aryloxy, N-sulfonylcarboxyamido, N-acylamino, hydroxy, aryl, andcycloalkyl, —O(CH₂)_(n)CO₂R¹⁷, —O(CH₂)_(n)CONHSO₂R¹⁸, —(CH₂)_(n)CO₂R¹⁹,—(CH₂)_(n)CONHSO₂R²⁰, —(O)NHCH(R²²)CO₂R²¹, and —N(R²³)(CH₂)_(n)CO₂R²⁴;alternatively, each of R³ and R⁴ together, R⁵ and R⁶ together, and R⁷and R⁸ together are independently selected from the group consisting of

wherein each of R¹⁴, R¹⁵ and R¹⁶ are independently selected from thegroup consisting of hydrido, alkyl and carboxy substituted alkyl;provided that at least five of R³, R⁴, R⁵, R⁶, R⁷, and R⁸ areindependently hydrido; (e) wherein Hat is selected from the groupconsisting of

wherein X is selected from the group consisting of N and CR²⁷; wherein Yis selected from the group consisting of NH, S and O; wherein Z isselected from the group consisting of N and CR²⁸; wherein each of R²⁵,R²⁶, R²⁷, and R²⁸ is independently selected from the group consisting ofnitro, halo, hydroxy, lower amino, lower alkyl, lower alkoxy, lowercarboalkoxy and carboxy; and pharmaceutically-acceptable salts thereof;and wherein each of R²⁹, R³⁰, and R³¹ is selected from the groupconsisting of hydrido, alkyl aryl, nitro and amino.
 2. The compound ofclaim 1 wherein Ar is aryl and pharmaceutically-acceptable saltsthereof.
 3. The compound of claim 1 wherein L is —C(O)NHCH₂— andpharmaceutically-acceptable salts thereof.
 4. The compound of claim 1wherein each of R¹, R², R⁹, R¹⁰, R¹¹, R¹² and R¹³ is hydrido andpharmaceutically-acceptable salts thereof.
 5. The compound of claim 1wherein each of R³, R⁴, R⁵, R⁶, R⁷, and R⁸ is independently selectedfrom the group consisting of hydrido, hydroxy, alkoxy, alkyl, amino, andcarboxyamido; and pharmaceutically-acceptable salts thereof.
 6. Thecompound of claim 1 wherein each of R³, R⁴, R⁵, R⁶, R⁷, and R⁸ isindependently selected from the group consisting of hydrido,—O(CH₂)_(n)CO₂R¹⁷, —O(CH₂)_(n)CONHSO₂R¹⁸, —(CH₂)_(n)CO₂R¹⁹,—(CH₂)_(n)CONHSO₂R²⁰, —C(O)NHCH(R²²)CO₂R²¹, and —N(R²³)(CH₂)_(n)CO₂R²⁴;wherein each of R¹⁷, R¹⁹, R²¹, R²², R²³, and R²⁴ is independentlyselected from the group consisting of hydrido and alkyl; wherein R¹⁸ andR²⁰ are independently alkyl; wherein n is selected from the groupconsisting of 1 and 2; and pharmaceutically-acceptable salts thereof. 7.The compound of claim 6 wherein R³, R⁴, R⁶, R⁷, and R⁸ are hydrido andR⁵ is selected from the group consisting of —O(CH₂)_(n)CO₂R¹⁷,—O(CH₂)_(n)CONHSO₂R¹⁸, —(CH₂)_(n)CO₂R¹⁹, —(CH₂)_(n)CONHSO₂R²⁰,—C(O)NHCH(R²²)CO₂R²¹, and —N(R²³)(CH₂)_(n)CO₂R²⁴ andpharmaceutically-acceptable salts thereof.
 8. The compound of claim 1wherein Hat is selected from the group consisting of

and pharmaceutically-acceptable salts thereof.
 9. A compound of claim 1of the Formula:

and pharmaceutically-acceptable salts thereof.
 10. A pharmaceuticalcomposition comprising a therapeutically-effective amount of an activecompound and a pharmaceutically-acceptable carrier, said active compoundselected from a family of compounds of the Formula:

(a) wherein Ar is selected from the group consisting of substituted orunsubstituted aryl, and substituted or unsubstituted heteroaryl; (b)wherein L is selected from the group consisting of —C(O)N(Q)CH₂—, and—CR¹⁰R¹¹OCR¹²R¹³—; wherein Q is selected from the group consisting ofhydrido, —(CH₂)_(m)CO₂H and —(CH₂)_(m)CO₂CH₃; wherein m is selected fromthe group consisting of 1, 2, 3, and 4; (c) wherein each of R¹, R², R⁹,R¹⁰, R¹¹, R¹² and R¹³ is independently selected from the groupconsisting of hydrido and lower alkyl; (d) wherein each of R³, R⁴, R⁵,R⁶, R⁷, and R⁸ is independently selected from the group consisting ofhydrido, acyl, amino, cyano, acyloxy, acylamino, carboalkoxy,carboxyamido, carboxy, halo, alkyl, heteroaryl, heterocyclyl, alkoxy,aryloxy, N-sulfonylcarboxyamido, N-acylamino, hydroxy, aryl, andcycloalkyl, —O(CH₂)_(n)CO₂R¹⁷, —O(CH₂)_(n)CONHSO₂R¹⁸, —(CH₂)_(n)CO₂R¹⁹,—(CH₂)_(n)CONHSO₂R²⁰, —C(O)NHCH(R²²)CO₂R²¹, and —N(R²³)(CH₂)_(n)CO₂R²⁴;alternatively, each of R³ and R⁴ together, R⁵ and R⁶ together, and R⁷and R⁸ together are independently selected from the group consisting of

wherein each of R¹⁴, R¹⁵ and R¹⁶ are independently selected from thegroup consisting of hydrido, alkyl and carboxy substituted alkyl;provided that at least five of R³, R⁴, R⁵, R⁶, R⁷, and R⁸ areindependently hydrido; (e) wherein Hat is selected from the groupconsisting of

wherein X is selected from the group consisting of N and CR²⁷; wherein Yis selected from the group consisting of NH, S and O; wherein Z isselected from the group consisting of N and CR²⁸; wherein each of R²⁵,R²⁶, R²⁷, and R²⁸ is independently selected from the group consisting ofnitro, halo, hydroxy, lower amino, lower alkyl, lower alkoxy, lowercarboalkoxy and carboxy; wherein each of R²⁹, R³⁰, and R³¹ is selectedfrom the group consisting of hydrido, alkyl, aryl, nitro and amino; andpharmaceutically-acceptable salts thereof.
 11. The composition of claim10 wherein Ar is aryl and pharmaceutically-acceptable salts thereof. 12.The composition of claim 10 wherein L is —C(O)NHCH₂— andpharmaceutically-acceptable salts thereof.
 13. The composition of claim10 wherein each of R¹, R², R⁹, R¹⁰, R¹¹, R¹² and R¹³ is independentlyselected from the group consisting of hydrido andpharmaceutically-acceptable salts thereof.
 14. The composition of claim10 wherein each of R³, R⁴, R⁵, R⁶, R⁷, and R⁸ is independently selectedfrom the group consisting of hydrido, hydroxy, alkoxy, alkyl, amino, andcarboxyamido; and pharmaceutically-acceptable salts thereof.
 15. Thecomposition of claim 10 wherein each of R³, R⁴, R⁵, R⁶, R⁷, and R⁸ isindependently selected from the group consisting of hydrido,—O(CH₂)_(n)CO₂R¹⁷, —O(CH₂)_(n)CONHSO₂R¹⁸, —(CH₂)_(n)CO₂R¹⁹,—(CH₂)_(n)CONHSO₂R²⁰, —C(O)NHCH(R²²)CO₂R²¹, and —N(R²³)(CH₂)_(n)CO₂R²⁴;wherein each of R¹⁷, R¹⁹, R²¹, R²², R²³, and R²⁴ is independentlyselected from the group consisting of hydrido and alkyl; wherein R¹⁸ andR²⁰ are independently selected from the group consisting of alkyl;wherein n is selected from the group consisting of 1 and 2; andpharmaceutically-acceptable salts thereof.
 16. The composition of claim15 wherein R³, R⁴, R⁶, R⁷, and R⁸ are hydrido and R⁵ is selected fromthe group consisting of —O(CH₂)_(n)CO₂R¹⁷, —O(CH₂)_(n)CONHSO₂R¹⁸,—(CH₂)_(n)CO₂R¹⁹, —(CH₂)_(n)CONHSO₂R²⁰, —C(O)NHCH(R²²)CO₂R²¹, and—N(R²³)(CH₂)_(n)CO₂R²⁴ and pharmaceutically-acceptable salts thereof.17. The composition of claim 1 wherein Hat is

and pharmaceutically-acceptable salts thereof.
 18. The composition ofclaim 10 wherein said active compound is selected from a family ofcompounds of the Formula:

and pharmaceutically-acceptable salts thereof.
 19. A compound of theformula

selected from the Table L R₅ N (S)—CONHCH₂ H 1 (S)—CONHCH₂ (R,S) OH 1(S)—CONHCH₂ (R,S)—CN 1 (S)—CONHCH₂ (R,S)—OCH₂Ph 1 (S)—CONHCH₂ ═O 1(S)—CONHCH₂ H 2 (S)—CONHCH₂ ═NNH₂ 1 (S)—CONHCH₂ (R,S)-tetrazole 1(S)—CONHCH₂ ═NOH 1 (S)—CONHCH₂ ═NOCH₃ 1 (S)—CONHCH₂ ═NOCH₂CO₂H 1(R)-CONHCH₂ H 1 (R)-CONHCH₂ (R)-OH 1 (S)—CONHCH₂ (S)—OH 1 (S)—CONHCH₂(R)-OH 1 (S)—CH₂OCH₂ H 1 (S)—CONHCH₂ CH₂CO₂H 1


20. A compound selected from the group consisting of


21. A compound selected from the group consisting of


22. A compound selected from the group consisting of


23. A compound selected from the group consisting of


24. A compound selected from the group consisting of


25. A compound selected from the group consisting of


26. A compound selected from the group consisting of


27. A compound selected from the group consisting of


28. A compound selected from the group consisting of


29. A compound selected from the group consisting of


30. A compound selected from the group consisting of


31. A compound selected from the group consisting of


32. A compound of the formula


33. A compound of the formula


34. A compound of the formula


35. A compound of the formula


36. A compound of the formula


37. A compound of the formula